DNA fragments -0.57, -2.2, -2.9, -5.3, and -8.4 kb in length from the regulatory region of the vnd/NK-2 gene were cloned in the 5'- flanking region of a beta-galactosidase(b-gal) reporter gene in the P-element pCaSpeR-AUG-b-gal, and the effects of the DNA inserts on the pattern and time of expression of b-gal were determined in transgenic Drosophila embryos. The results showed that the -8.4 kb DNA fragment contains the nucleotide sequences required to generate the normal pattern of vnd/NK-2 gene expression, and that -5.3 to -8.4 kb DNA is required for vnd/NK-2 gene expression in the most dorsoanterior part of the cephalic region. No partial pattern of vnd/NK-2 gene expression was detected with smaller fragments of DNA tested. vnd/NK-2 mutant embryos containing the homozygous P-element p[-8.4 to + 0.34 b-gal] expressed little or no beta-gal in contrast to embryos with a wild-type vnd/NK-2 gene. Therefore, vnd/NK-2 protein is required, directly or indirectly, for maintenance of vnd/NK-2 gene expression. In a collaborative study with James Ferretti and his associates, the three dimensional structure of the vnd/NK-2 homeodomain was determined. The effects of mutations in positions 52, 54, and 56 of the vnd/NK-2 homeodomain on the secondary structure of the homeodomain and the ability of the homeodomain protein to bind to DNA were determined. Three single sites H52R, Y54M, and T56W mutations, 2 double site H52R/T56W and Y54M/T56W mutations, and one triple site H52R/Y54M/T56W mutations were investigated. Replacement of Y54 with M resulted in a ten- fold decrease in the affinity of the homeodomain for DNA containing the vnd/NK-2 consensus sequence, CAAGTG. T56 plays an important role in determining the length of the DNA recognition helix. Replacement of H52 with R does not affect the length of the helix but does increase the thermal stability. Replacement of A35 by T is a lethal mutation in Drosophila. The mutant homeodomain was shown to be unable to adopt a folded conformation. The affinity of the A35T mutant vnd/NK-2 homeodomain for the vnd/NK-2 target DNA sequence is 50-fold lower than that of the wild-type homeodomain.